Amyloid Beta (Aβ) peptides are generated from the cleavage of Amyloid Precursor Protein (APP) via beta secretase and gamma secretase enzymatic complexes (Wolfe, Biochemistry, 45:7931-7939 (2006)). Beta secretase generates the N-terminal ends of these amyloid peptides and gamma secretase generates the C-terminal ends (Wolfe, Biochemistry, 45:7931-7939 (2006)). Several species of peptides are subsequently generated, typically ranging from 38 to 42 amino acids in length depending on where the gamma secretase cleaves the APP. Aβ peptides have an extracellular domain (amino acids 1-28) and a transmembrane region (amino acids 29-42) that is embedded in the lipid bilayer. Amyloid peptides that are 42 amino acids long (Aβ42) are believed to be the putative neurotoxic species, either alone or as aggregates. These aggregates are suspected to contribute to the neurodegeneration of the brain resulting in Alzheimer's disease and dementia. The hypothesis that Aβ42 contributes to clinical dementias is called the amyloid cascade hypothesis as described by Hardy et al. (Science, 256: 184-185 (1992)).
One of the characteristics of the AP peptides is the ability to self-assemble into oligomers at physiological concentrations (Burdick et al, Journal of Biological Chemistry, 267:546-554 (1992); Cerf et al, Biochemical Journal, 421:415-423 (2009)). The Aβ42 species is more prone to forming oligomers as compared to the Aβ40 and Aβ38 species. The mechanism of oligomer formation has been shown to originate from a small, five amino acid region located at amino acids 16 to 20 (KLVFF) which mediates the binding of Aβ peptides in an anti-parallel manner. This small region has thus been termed the “aggregation domain” (Tjernberg et al, Journal of Biological Chemistry, 271:8545-8548 (1996)). Aβ peptide aggregates assemble rapidly (i.e., within minutes) under certain conditions especially at lower pH ranges, with slightly slower kinetics at neutral or higher pH (Burdick et al, Journal of Biological Chemistry, 267:546-554 (1992)). The aggregates are poorly soluble in aqueous solutions, especially in the presence of salts. The C-terminal ends of the Aβ peptides fold back over the core of the dimer via salt bridges thereby increasing their hydrophobicity and promoting further polymerization of the peptides into filaments or fibrils. The additional two C-terminal residues in the Aβ42 species provides increased hydrophobicity in comparison to the other Aβ species (Kim et al, Journal of Biological Chemistry, 280:35069-35076 (2005)).
Clinical data suggests that the degree of dementia and cognitive decline has a higher correlation with Aβ42 concentration than either the Aβ4o or Aβ38 species. This observation, in conjunction with the rapid aggregation properties of Aβ42, has led to the hypothesis that inhibition of Aβ42 aggregation may have clinical benefits. There have been numerous studies showing different mechanisms that can be used to inhibit the formation of Aβ42 aggregates. Tjernberg et al. (Journal of Biological Chemistry, 271: 8545-8548 (1996)) showed that peptides comprising the aggregation domain bind well to Aβ peptides and inhibit the formation of aggregates. Several other molecules that bind to the aggregation domain have also been shown to inhibit amyloid peptide aggregation (Martharu et al, Journal of Neurological Sciences, 280:49-58 (2009); Kim et al, Biochemical and Biophysical Research Communications, 303:576-279 (2003)). Substitution of amino acids in the aggregation core domain or the deletion of the entire aggregation domain also prevents Aβ peptide aggregation and fibril formation (Tjernberg et al, Journal of Biological Chemistry, 274: 12619-12625 (1999)). In addition, various drugs have been designed to inhibit gamma secretase activity in order to lower the amount of Aβ42 and related peptide species. The usefulness of these approaches in the clinic is currently under investigation.
In order to assess the effectiveness of a molecule to inhibit the generation of Aβ42 or to prevent its aggregation, it is necessary to measure the amount of Aβ42 accurately. There are several techniques that are used to detect and quantitate Aβ42 in biological samples including both immunoassays (Olsson et al, Clinical Chemistry, 51:336-345 (2005); Verwey et al, Journal of Immunological Methods, 348:57-66 (2009); Sjogren et al, Journal of Neural Transmission, 107:563-679 (2000)) and mass spectrometry (MS) based methods (Cantone et al, Journal of Neuroscience Methods, 180:255-260 (2009); Journal of Mass Spectrometry, 40: 142-145 (2005)). The MS based methods, including those of MALDI-TOF and SELDI-TOF along with liquid chromatography prepared mass spectrometry, are able to detect many of the amyloid beta species in a biological sample, but do not presently provide sufficient quantitative values that are needed for measuring Aβ42 in clinical samples.
Immunoassay methods are based on a double sandwich immunoassay that comprises one antibody that is specific to the N-terminus and a second antibody that is highly specific for the Aβ42 C-terminus (i.e., does not recognize other Aβ peptide species). There are two basic versions of the immunoassays. The first version captures Aβ peptides in biological samples via a solid surface immobilized N-terminal region specific antibody. The Aβ42 specific antibody carrying a tag is added to the immunoassay in order to complete the antibody sandwich. The second version captures Aβ peptides in biological samples via an Aβ42 C-terminal region specific antibody immobilized on a solid surface. The N-terminal region specific antibody carrying a tag is added to the immunoassay. In either version, the tag incorporated via the second antibody enables the detection of the complete complex. These assays are made quantitative by the use of Aβ42 reference standards, which are added in lieu of biological samples. The resulting signal measured from the reference standards are used to generate a standard curve which is subsequently used to quantify the amount of Aβ42 in the biological samples.
Until now, the use of Aβ42 reference standards in immunoassays has relied on synthetic, full length Aβ42 peptides which are typically generated with minimal difficulty. However, these peptides have strong hydrophobic properties and, therefore, are not soluble in aqueous solutions. In addition, the storage and use of Aβ42 as reference standards presents many issues. As discussed, Aβ42 forms aggregates rapidly and this formation occurs more readily at room temperatures and neutral pH. Long term storage at low temperatures (below −20° C.) and low pH helps to minimize aggregation during storage but it does not prevent it. Reconstitution of Aβ42 in buffers that are amenable to immunoassays can also prove difficult. These solutions are almost always aqueous, buffered at a neutral pH, contain salts, and used at room temperature; all the conditions that accelerate Aβ42 aggregation. Aβ42 peptides that have aggregated are not useful as reference standards in immunoassays because of the insoluble precipitates and non-uniformity in both size and availability to be recognized by either detection or capture antibodies.
Thus, the present invention fulfills a need in the art by providing methods useful for generating an Aβ42 peptide or protein construct and compositions thereof that can be used as a reference standard or calibrator in an immunoassay or other format to measure the abundance of Aβ42 peptide accurately in a fluid or tissue extract sample. Specifically, the compositions and methods of the present invention are aimed at creating non-aggregating peptide reference standards for Aβ42 for use in immunoassay formats. The compositions and methods described herein have a broad applicability to many other peptides that are difficult to measure and quantitate.